Vitamin C (L-ascorbic acid or L-ascorbate) is an essential co-factor for enzymes catalyzing numerous biochemical reactions including hydroxylation, as well as a primary antioxidant in both plants and animals. In plants, L-ascorbate has been implicated in processes including growth (Pignocchi and Foyer, 2003, Curr. Opin. Plant Biol. 6, 379-389) programmed cell death (de Pinto et al., 2006, Plant J. 48, 784-795), pathogen responses (Barth et al., 2004, Plant Physiol. 134, 1784-1792), hormone responses, flowering and senescence (Barth et al., 2006, J. Exp. Bot. 57, 1657-1665), as well as protection against environmental stresses including ozone (Conklin and Barth, 2004, Plant Cell Environ. 27, 959-970), UV radiation (Gao and Zhang, 2008, J. Plant Physiol. 165, 138-148), high temperatures (Larkindale et al., 2005, Plant Physiol. 138, 882-897) and high light intensity (Muller-Moulé et al. 2004, Plant Physiol. 134, 1163-1172).
The concentration in ascorbate in plants and plant cells is determined, on the one hand by de novo ascorbate synthesis as well as regeneration of ascorbate from its oxidized forms, and on the other hand, by consumption of ascorbate in the detoxification of reactive oxygen species (ROS) and hydrogenperoxide (H2O2) generated through the respiration process or in response to stress conditions.
Synthesis of vitamin C in plants has only been relatively recently elucidated and is referred to as the L-galactose pathway or Smirnoff-Wheeler pathway (Smirnoff et al. 2000, Crit. Rev. Biochem. Mol. Biol. 35, 291-314). The first six steps of the L-galactose pathway synthesize activated nucleotide sugars that are also precursors of cell wall polysaccharides and glycoproteins. The committed pathway to L-ascorbate biosynthesis then consists of the sequential conversion of GDP-L-galactose into L-galactose-1-P, L-galactose, L-galactono-1,4-lactone and L-ascorbate. The enzyme catalyzing the last step reaction, L-galactono-1,4 lactone dehydrogenase, is associated with mitochondrial NADH-ubiquinone oxidoreductase (complex I) (Pineau et al., 2008, J. Biol. Chem. 283, 32500-32505).
Ascorbate becomes rapidly oxidized to monodehydroascorbate via reactions involving oxidative species (including reduction of H2O2 through ascorbate peroxidase). Monodehydroascorbate is further oxidized spontaneously to dehydroascorbate. To prevent the degradation of dehydroascorbate via ring opening, dehydroascorbate must be rapidly recycled to avoid depletion of the ascorbate pools.
Ascorbate recycling also occurs in the plant mitochondria. Dehydroascorbate can be reduced to ascorbate by two main mechanisms. Electrons can be provided by small electron carriers, such as glutathione or lipoic acid, through the action of dehydroascorbate reductase, or by the respiratory electron transfer chain. Using substrates and inhibitors of the respiratory electron transfer chain, the site of dehydroascorbate reduction was localized to complex II (Szarka et al. 2007, Physiologia Plantarum 129: 225-232).
Talla et al. (2011, J. BioSci 36, 163-173) suggest that ascorbic acid is a key participant during the interactions between chloroplasts and mitochondria to optimize photosynthesis and protect against photoinhibition.
The currently used methods to determine the yield potential of plant lines (such as plant lines resulting from conventional breeding activities, and/or plant lines with an engineered trait, be it through transgenesis, mutagenesis or other means) consists in performing field trials at different locations, preferably under different conditions. A disadvantage of field trials for crop plants, is that, at best, only two experiments can be done each year. Even when field trials are planned very well and deliver the appropriate data, this time constraint interferes with the continuity of the projects and slows down the progress.
A number of assays (mainly qualitative) have been described for use in plant tissue culture to study the effect of various stresses on the survival of cells or tissues (Towill and Mazur, 1975; Chen et al. 1982, Duncan and Widholm 1990, Stepan-Sarkissian and Grey, 1990; Upadhyaya and Caldwell, 1993; Enikeev et al., 1995; Ishikawa et al., 1995; Popov and Vysotskaya, 1996). These are actually “viability” assays which do not measure the yield potential of plants.
Chlorophyll fluorescence and fluorescence imaging may also be used to study the influences of stress conditions on whole plants (Lichtenthaler, 1996; Lichtentaler and Mieké, 1997). Although these assays provide some data on the tolerance of the plant lines to certain stresses, they cannot be used to measure yield potential.
WO 97/06267 describe the use of PARP inhibitors to improve the transformation (qualitatively or quantitatively) of eukaryotic cells, particularly plant cells. Also described is a method for assessing the agronomical fitness of plants or plant material by measuring the electron flow in the mitochondrial electron transport chain.
WO 2002/066972 provides methods and means for determining parent inbred plant lines with good combining ability, for determining good combinations of parent inbred plant lines capable of yielding hybrid lines with high heterosis, and further for determining the agronomical performance of different plant lines, which can be performed in vitro by determining the electron flow in the mitochondria under control and stress conditions.
There remains however a need to improve the prediction methods described in the art to arrive at an improved method allowing to predict important plant characteristics, such as yield potential or increased shelf life, in an early stage of plant development, with sufficient accuracy, without having to resort to field trials. Such methods would represent an extra tool to rapidly identify plant lines of high interest in breeding programs, allowing to discard non-promising lines rapidly and could result in a significant gain of time and resources.
The current invention provides such methods as described in the various embodiments and claims disclosed herein.